Dihydropteridione derivatives, process for their manufacture and their use as medicament

ABSTRACT

Disclosed are new dihydropteridinones of the formula (I) 
                         
wherein the groups L, R 1 , R 2 , R 3 , R 4  and R 5  have the meanings provided herein, the isomers thereof, processes for preparing these dihydropteridinones and their use as pharmaceutical compositions.

APPLICATION DATA

This application is a divisional application of U.S. application Ser.No. 11/210,379 filed on Aug. 24, 2005 which claims benefit to EuropeanPatent Application no. EP 04 020 124.6 filed Aug. 25, 2004.

FIELD OF INVENTION

The present invention relates to new dihydropteridinones of generalformula (I)

wherein the groups L, R¹, R², R³, R⁴ and R⁵ have the meanings given inthe claims and specification, the isomers thereof, processes forpreparing these dihydropteridinones and their use as pharmaceuticalcompositions.

BACKGROUND TO THE INVENTION

Pteridinone derivatives are known from the prior art as activesubstances with an antiproliferative activity. WO 01/019825 and WO03/020722 describe the use of pteridinone derivatives for the treatmentof tumoral diseases.

Tumour cells wholly or partly elude regulation and control by the bodyand are characterised by uncontrolled growth. This is based on the onehand on the loss of control proteins, such as e.g. Rb, p16, p21 and p53and also on the activation of so-called accelerators of the cell cycle,the cyclin-dependent kinases (CDK's).

In addition, the protein kinase Aurora B has been described as having anessential function during entry into mitosis. Aurora B phosphorylateshistone H3 at Ser10 and thus initiates chromosome condensation (Hsu etal. 2000, Cell 102:279-91). A specific cell cycle arrest in the G2/Mphase may however also be triggered e.g. by the inhibition of specificphosphatases such as e.g. Cdc25C (Russell and Nurse 1986, Cell45:145-53). Yeasts with a defective Cdc25 gene arrest in the G2 phase,while overexpression of Cdc25 leads to premature entry into the mitosisphase (Russell and Nurse 1987, Cell 49:559-67). Moreover, an arrest inthe G2/M phase may also be triggered by the inhibition of certain motorproteins, the so-called kinesins such as e.g. Eg5 (Mayer et al. 1999,Science 286:971-4), or by agents which stabilise or destabilisemicrotubules (e.g. colchicin, taxol, etoposide, vinblastin, vincristine)(Schiff and Horwitz 1980, Proc Natl Acad Sci USA 77:1561-5).

In addition to the cyclin-dependent and Aurora kinases the so-calledpolo-like kinases, a small family of serine/threonine kinases, play animportant part in the regulation of the eukaryotic cell cycle. Hitherto,the polo-like kinases PLK-1, PLK-2, PLK-3 and PLK-4 have been describedin the literature. PLK-1 in particular has been shown to play a centralpart in the regulation of the mitosis phase. PLK-1 is responsible forthe maturation of the centrosomes, for the activation of phosphataseCdc25C, and for the activation of the Anaphase Promoting Complex (Gloveret al. 1998, Genes Dev. 12:3777-87; Qian et al. 2001, Mol Biol Cell.12:1791-9). The injection of PLK-1 antibodies leads to a G2 arrest inuntransformed cells, whereas tumour cells arrest in the mitosis phase(Lane and Nigg 1996, J Cell Biol. 135:1701-13). Overexpression of PLK-1has been demonstrated for various types of tumour, such asnon-small-cell lung cancer, plate epithelial carcinoma, breast andcolorectal carcinoma (Wolf et al. 1997, Oncogene 14:543-549; Knecht etal. 1999, Cancer Res. 59:2794-2797; Wolf et al. 2000, Pathol. Res.Pract. 196:753-759; Takahashi et al. 2003, Cancer Sci. 94:148-52).Therefore, this category of proteins also constitutes an interestingapproach to therapeutic intervention in proliferative diseases (Liu andErikson 2003, Proc Natl Acad Sci USA 100:5789-5794).

The resistance of many types of tumours calls for the development of newpharmaceutical compositions for combating tumours.

The aim of the present invention is to provide new compounds having anantiproliferative activity.

DESCRIPTION OF THE INVENTION

The problem according to the invention is solved by the followingcompounds of formula (I).

Accordingly, the present invention relates to dihydropteridinones ofgeneral formula (I)

wherein

-   L denotes a single bond, or a bridging double-bonded group selected    from among C₁-C₆-alkylene, C₂-C₆-alkenylene, C₂-C₆-alkynylene,    C₃-C₇-cycloalkylene, C₁-C₄-alkylene-C₆-C₁₀-arylene-C₁-C₄-alkylene,    C₁-C₄-alkylene-C₆-C₁₀-arylene, —O, —O—C₁-C₆-alkylene,    —O—C₃-C₆-alkenylene, —O—C₃-C₆-alkynylene, —O—C₃-C₇-cycloalkylene,    —O—C₁-C₄-alkylene-C₆-C₁₀-arylene-C₁-C₄-alkylene,    —O—C₁-C₄-alkylene-C₆-C₁₀-arylene, —NR⁷— and —NR⁷—C₁-C₆-alkylene,    —NR⁷—C₃-C₆-alkenylene, —NR⁷—C₃-C₆-alkynylene,    —NR⁷—C₃-C₇-cycloalkylene,    —NR⁷—C₁-C₄-alkylene-C₆-C₁₀-arylene-C₁-C₄-alkylene,    —NR⁷—C₁-C₄-alkylene-C₆-C₁₀-arylene, which may optionally be    substituted by one or more groups R⁹;-   R¹ and R², which may be identical or different, denote hydrogen, or    a group selected from among C₁-C₆-alkyl, C₂-C₆-alkenyl and    C₂-C₆-alkynyl, which may optionally be mono- or polysubstituted by    one or more groups R⁹, or-   R¹ and R² together denote C₂-C₆-alkylene, in which optionally one or    two methylene groups may be replaced by one of the groups —O or    —NR⁷, and which may optionally be mono- or polysubstituted by one or    more groups R⁹;-   R³ denotes hydrogen or a group selected from C₁-C₈-alkyl,    C₂-C₈-alkenyl, C₂-C₈-alkynyl, C₃-C₈-cycloalkyl and C₆-C₁₄-aryl,    which may optionally be mono- or polysubstituted by one or more    groups R⁹; or-   R¹ and R² or R³ and R¹ together denote C₂-C₆-alkylene which may    optionally be mono- or polysubstituted by one or more groups R⁹;-   R⁴ denotes hydrogen, halogen, CN, OH, —NR⁷R⁸ or a group selected    from among C₁-C₆-alkyl, C₁-C₆-alkyloxy, C₂-C₆-alkenyl,    C₂-C₆-alkenyloxy, C₂-C₆-alkynyl and C₂-C₆-alkynyloxy, which may    optionally be mono- or polysubstituted by one or more groups R¹⁰;-   R⁵ denotes C₃-C₈-cycloalkyl which may optionally be mono- or    polysubstituted by one or more groups R⁶, or-   R⁵ denotes C₃-C₈-cycloalkyl which may optionally be mono- or    polysubstituted by one or more groups R⁹, or-   R⁵ denotes a 5-10-membered heteroaryl group which may contain one,    two or three heteroatoms selected from among nitrogen, oxygen and    sulphur, preferably nitrogen or oxygen, and which may optionally be    mono- or polysubstituted by one or more of the groups R¹¹, or-   R⁵ denotes a 5-10-membered heterocycloalkyl group which may contain    one, two or three heteroatoms selected from among nitrogen, oxygen    and sulphur, preferably nitrogen or oxygen, and which may optionally    be mono- or polysubstituted by one or more of the groups R¹¹;-   R⁶ denotes —NR⁷R⁸ or a 5-10-membered heterocycloalkyl group which    may contain one, two or three heteroatoms selected from among    nitrogen, oxygen and sulphur, preferably nitrogen or oxygen, and    which may optionally be mono- or polysubstituted by one or more of    the groups R¹²;-   R⁷ and R⁸, which may be identical or different, denote hydrogen or    C₁-C₆-alkyl,-   R⁹ denotes halogen, CN, OH or CF₃;-   R¹⁰ denotes halogen, OH, CN, ═O, C₁-C₆-alkyloxy, COOR⁷, NR⁷R⁸,    CONR⁷R⁸, SO₂R⁷, CHF₂ or CF₃;-   R¹¹ denotes halogen, C₁-C₄-alkyl, OH, CF₃, C₆-C₁₀-aryl or    C₁-C₆-alkylene-C₆-C₁₀-aryl;-   R¹² denotes halogen, CF₃, C₁-C₆-alkyl, C₃-C₈-cycloalkyl or    C₁-C₆-alkylene-C₃-C₈-cycloalkyl;    optionally in the form of the tautomers, racemates, enantiomers,    diastereomers and mixtures thereof, and optionally in the form of    the pharmacologically acceptable acid addition salts, solvates    and/or hydrates thereof.

Preferred are compounds of general formula (I), wherein

-   L denotes a single bond, or a bridging double-bonded group selected    from among C₁-C₆-alkylene, C₂-C₆-alkenylene, C₂-C₆-alkynylene,    C₃-C₇-cycloalkylene, C₁-C₄-alkylene-C₆-C₁₀-arylene-C₁-C₄-alkylene,    —O, —O—C₁-C₄-alkylene, —NR⁷— and —NR⁷—C₁-C₄-alkylene, which may    optionally be substituted by one or more groups R⁹;-   R¹ and R², which may be identical or different, denote hydrogen, or    a group selected from among C₁-C₆-alkyl, C₂-C₆-alkenyl and    C₂-C₆-alkynyl, which may optionally be mono- or polysubstituted by    one or more groups R⁹, or-   R¹ and R² together denote C₂-C₆-alkylene, in which optionally one or    two methylene groups may be replaced by one of the groups —O or    —NR⁷, and which may optionally be mono- or polysubstituted by one or    more groups R⁹;-   R³ denotes hydrogen or a group selected from C₁-C₈-alkyl,    C₂-C₈-alkenyl, C₂-C₈-alkynyl, C₃-C₈-cycloalkyl and C₆-C₁₄-aryl,    which may optionally be mono- or polysubstituted by one or more    groups R⁹; or-   R³ and R² or R³ and R¹ together denote C₂-C₆-alkylene which may    optionally be mono- or polysubstituted by one or more groups R⁹;-   R⁴ denotes hydrogen, halogen, CN, OH, —NR⁷R⁸ or a group selected    from among C₁-C₆-alkyl, C₁-C₆-alkyloxy, C₂-C₆-alkenyl,    C₂-C₆-alkenyloxy, C₂-C₆-alkynyl and C₂-C₆-alkynyloxy, which may    optionally be mono- or polysubstituted by one or more groups R¹⁰;-   R⁵ denotes C₃-C₈-cycloalkyl, which may optionally be substituted by    a group R⁶, or-   R⁵ denotes C₃-C₈-cycloalkyl which may optionally be mono- or    polysubstituted by one or more groups R⁹, or-   R⁵ denotes a 5-10-membered heteroaryl group which may contain one,    two or three heteroatoms selected from among nitrogen, oxygen and    sulphur, preferably nitrogen or oxygen, and which may optionally be    mono- or polysubstituted by one or more of the groups R⁹, or-   R⁵ denotes a 5-10-membered heterocycloalkyl group which may contain    one, two or three heteroatoms selected from among nitrogen, oxygen    and sulphur, preferably nitrogen or oxygen, and which may optionally    be mono- or polysubstituted by one or more of the groups R¹¹;-   R⁶ denotes —NR⁷R⁸ or a 5-10-membered heterocycloalkyl group which    may contain one, two or three heteroatoms selected from among    nitrogen, oxygen and sulphur, preferably nitrogen or oxygen, and    which may optionally be mono- or polysubstituted by one or more of    the groups R¹²;-   R⁷ and R⁸, which may be identical or different, denote hydrogen or    C₁-C₆-alkyl,-   R⁹ denotes halogen, CN, OH or CF₃;-   R¹⁰ denotes halogen, OH, CN, ═O, C₁-C₆-alkyloxy, COOR⁷, CONR⁷R⁸,    SO₂R⁷, CHF₂ or CF₃;-   R¹¹ denotes halogen, C₁-C₄-alkyl, OH, CF₃, C₆-C₁₀-aryl or    C₁-C₆-alkylene-C₆-C₁₀-aryl;-   R¹² denotes halogen, CF₃, C₁-C₆-alkyl, C₃-C₈-cycloalkyl or    C₁-C₆-alkylene-C₃-C₈-cycloalkyl;    optionally in the form of the tautomers, racemates, enantiomers,    diastereomers and mixtures thereof, and optionally in the form of    the pharmacologically acceptable acid addition salts, solvates    and/or hydrates thereof.

Also preferred are compounds of general formula (I), wherein

-   L denotes a single bond, —O, —O—C₁-C₃-alkylene, —NR⁷,    —NR⁷—C₁-C₃-alkylene or C₁-C₄-alkylene, which may optionally be    substituted by one or more groups R⁹;-   R¹ and R², which may be identical or different, denote hydrogen, or    a group selected from among C₁-C₄-alkyl, C₂-C₄-alkenyl and    C₂-C₄-alkynyl, which may optionally be mono- or polysubstituted by    one or more groups R⁹, or-   R¹ and R² together denote C₂-C₄-alkylene which may optionally be    mono- or polysubstituted by one or more groups R⁹;-   R³ denotes hydrogen or a group selected from C₁-C₆-alkyl,    C₃-C₇-cycloalkyl and C₆-C₁₀-aryl, which may optionally be mono- or    polysubstituted by one or more groups R⁹; or-   R³ and R² or R³ and R¹ together denote C₂-C₄-alkylene which may    optionally be mono- or polysubstituted by one or more groups R⁹;-   R⁴ denotes hydrogen, fluorine, chlorine, bromine, —NR⁷R⁸ or a group    selected from among C₁-C₄-alkyl, C₁-C₄-alkyloxy, C₂-C₄-alkenyloxy    and C₂-C₄-alkynyloxy, which may optionally be mono- or    polysubstituted by one or more groups R¹⁰;-   R⁵ denotes C₃-C₇-cycloalkyl, which may optionally be substituted by    a group R⁶, or-   R⁵ denotes C₃-C₇-cycloalkyl which may optionally be mono- or    polysubstituted by one or more groups R⁹, or-   R⁵ denotes a heteroaryl selected from among imidazolyl, oxazolyl,    isoxazolyl, pyrolyl, pyrazolyl, oxadiazolyl, pyridyl, pyridazinyl,    pyrazinyl, pyrimidinyl, indolyl, quinolinyl, isoquinolinyl,    benzimidazolyl, purinyl and pteridinyl, which may optionally be    mono- or polysubstituted by one or more of the groups R¹¹, or-   R⁵ denotes a heterocycloalkyl selected from among piperidinyl,    piperazinyl, pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl,    imidazolinyl, imidazolidinyl and morpholinyl which may optionally be    mono- or polysubstituted by one or more of the groups R¹¹;-   R⁶ denotes a heterocycloalkyl selected from among piperidinyl,    piperazinyl, pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl,    imidazolinyl, imidazolidinyl and morpholinyl which may optionally be    mono- or polysubstituted by one or more of the groups R¹²;-   R⁷ and R⁸, which may be identical or different, denote hydrogen or    C₁-C₄-alkyl,-   R⁹ denotes halogen, OH, ═O or CF₃;-   R¹⁰ denotes halogen, OH, ═O, C₁-C₄-alkyloxy or CF₃;-   R¹¹ denotes halogen, C₁-C₄-alkyl, OH, CF₃, phenyl or    C₁-C₄-alkylene-phenyl;-   R¹² denotes C₁-C₄-alkyl, C₃-C₆-cycloalkyl or    C₁-C₄-alkylene-C₃-C₆-cycloalkyl;    optionally in the form of the tautomers, racemates, enantiomers,    diastereomers and mixtures thereof, and optionally in the form of    the pharmacologically acceptable acid addition salts, solvates    and/or hydrates thereof.

Also preferred are compounds of general formula (I),

wherein

-   L denotes a single bond, C₁-C₄-alkylene, —O or NH—;-   R¹ and R², which may be identical or different, denote hydrogen, or    a group selected from among C₁-C₄-alkyl, C₂-C₄-alkenyl and    C₂-C₄-alkynyl, which may optionally be mono- or disubstituted by a    group selected from among fluorine, chlorine, OH and CF₃; or-   R³ denotes hydrogen or a group selected from C₁-C₆-alkyl,    C₃-C₇-cycloalkyl and C₆-C₁₀-aryl, which may optionally be mono- or    disubstituted by a group selected from among fluorine, chlorine, OH    and CF₃; or-   R⁴ denotes hydrogen, fluorine, chlorine, bromine, —NR⁷R⁸ or a group    selected from among C₁-C₄-alkyl, C₁-C₄-alkyloxy, C₂-C₄-alkenyloxy    and C₂-C₄-alkynyloxy, which may optionally be mono- or disubstituted    by a group selected from among fluorine, chlorine, OH, methoxy,    ethoxy and CF₃;-   R⁵ denotes C₃-C₇-cycloalkyl, which may optionally be mono- or    disubstituted by a group selected from among methyl, ethyl, propyl    OH, fluorine, chlorine, CF₃ and R⁶, or-   R⁵ denotes a heteroaryl selected from among imidazolyl, oxazolyl,    isoxazolyl, pyrolyl, pyrazolyl, oxadiazol, pyridyl, pyridazinyl,    pyrazinyl, pyrimidinyl, indolyl, quinolinyl, isoquinolinyl,    benzimidazolyl, purinyl and pteridinyl, which may optionally be    mono- or disubstituted, preferably monosubstituted, by fluorine,    chlorine, OH, CF₃, methyl, ethyl, propyl, phenyl, benzyl or    phenethyl,-   R⁵ denotes a heterocycloalkyl selected from among piperidinyl,    piperazinyl, pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl,    imidazolinyl, imidazolidinyl and morpholinyl, which may optionally    be mono- or disubstituted, preferably monosubstituted, by fluorine,    chlorine, OH, CF₃, methyl, ethyl, propyl, phenyl, benzyl or    phenethyl;-   R⁶ denotes a heterocycloalkyl selected from among piperidinyl,    piperazinyl, pyrrolinyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl,    imidazolinyl, imidazolidinyl and morpholinyl, which may be mono- or    disubstituted by methyl, ethyl, cyclopropyl or cyclopropylmethyl;-   R⁷ and R⁸, which may be identical or different, denote hydrogen or    C₁-C₄-alkyl, optionally in the form of the tautomers, racemates,    enantiomers, diastereomers and mixtures thereof, and optionally in    the form of the pharmacologically acceptable acid addition salts,    solvates and/or hydrates thereof.

Also preferred are compounds of general formula (I),

wherein

-   L denotes a single bond, —CH₂, —CH₂—CH₂, —O or NH—;-   R¹ and R², which may be identical or different, denote hydrogen or a    group selected from among methyl, ethyl, propyl, butyl, allyl and    propargyl, which may optionally be mono- or disubstituted by a group    selected from among fluorine, chlorine and CF₃;-   R³ denotes hydrogen or a group selected from among methyl, ethyl,    propyl, butyl, pentyl, cyclopropyl, cyclopentyl, cyclohexyl and    phenyl, which may optionally be mono- or disubstituted by a group    selected from among fluorine, chlorine and CF₃;-   R⁴ denotes hydrogen, methyl, ethyl, propyl, methyloxy, ethyloxy or    propyloxy;-   R⁵ denotes C₃-C₆-cycloalkyl, which may optionally be mono- or    disubstituted, preferably monosubstituted, by methyl, ethyl, propyl    OH, fluorine, chlorine, CF₃ or R⁶, or-   R⁵ denotes a heteroaryl selected from among pyridyl, pyrollyl and    pyrimidinyl, which may optionally be mono- or disubstituted,    preferably monosubstituted, by fluorine, chlorine, OH, CF₃, methyl,    ethyl, propyl, phenyl, benzyl or phenethyl,-   R⁵ denotes a heterocycloalkyl selected from among piperidinyl,    piperazinyl, pyrrolidinyl and morpholinyl, which may optionally be    mono- or disubstituted, preferably monosubstituted, by fluorine,    chlorine, OH, CF₃, methyl, ethyl, propyl, phenyl, benzyl or    phenethyl,-   R⁶ denotes a heterocycloalkyl selected from among piperidinyl,    morpholinyl, pyrrolidinyl and piperazinyl, which may optionally be    mono- or disubstituted by methyl, ethyl, cyclopropyl or    cyclopropylmethyl,    optionally in the form of the tautomers, racemates, enantiomers,    diastereomers and mixtures thereof, and optionally in the form of    the pharmacologically acceptable acid addition salts, solvates    and/or hydrates thereof.

Of particular importance according to the invention are compounds ofgeneral formula (I),

wherein

-   L denotes a single bond, or a bridging double-bonded group selected    from among C₁-C₄-alkylene, C₂-C₄-alkenylene, C₂-C₄-alkynylene,    C₃-C₆-cycloalkylene, —O, —O—C₁-C₃-alkylene, —NR⁷— and    —NR⁷—C₁-C₃-alkylene, which may optionally be substituted by one or    more groups R⁹,    and the groups R¹, R², R³, R⁴ and R⁵ and R⁹ may have one of the    meanings given above or hereinafter,    optionally in the form of the tautomers, racemates, enantiomers,    diastereomers and mixtures thereof, and optionally in the form of    the pharmacologically acceptable acid addition salts, solvates    and/or hydrates thereof.

Also of particular importance according to the invention are compoundsof general formula (I), wherein

-   L denotes a single bond, —O, —NH or C₁-C₄-alkylene, preferably a    single bond —NH, —CH₂ or —CH₂—CH₂, particularly preferably a single    bond or —NH—    and the groups R¹, R², R³, R⁴ and R⁵ may have one of the meanings    given above or hereinafter,    optionally in the form of the tautomers, racemates, enantiomers,    diastereomers and mixtures thereof, and optionally in the form of    the pharmacologically acceptable acid addition salts, solvates    and/or hydrates thereof.

Of particular importance according to the invention are compounds ofgeneral formula (I), wherein R¹ denotes hydrogen, methyl, ethyl, allylor propargyl, preferably hydrogen or methyl, particularly preferablyhydrogen and the groups L, R², R³, R⁴ and R⁵ may have one of themeanings given above or hereinafter, optionally in the form of thetautomers, racemates, enantiomers, diastereomers and mixtures thereof,and optionally in the form of the pharmacologically acceptable acidaddition salts, solvates and/or hydrates thereof.

Of particular importance according to the invention are compounds ofgeneral formula (I), wherein R² denotes hydrogen, methyl, ethyl, allylor propargyl, preferably methyl or ethyl, and the groups L, R¹, R³, R⁴and R⁵ may have one of the meanings given above or hereinafter,optionally in the form of the tautomers, racemates, enantiomers,diastereomers and mixtures thereof, and optionally in the form of thepharmacologically acceptable acid addition salts, solvates and/orhydrates thereof.

Of particular importance according to the invention are compounds ofgeneral formula (I),

wherein

-   R³ denotes ethyl, propyl, butyl, pentyl, cyclopropyl, cyclopentyl,    cyclohexyl or phenyl, preferably propyl, butyl, pentyl, cyclopentyl    or cyclohexyl, particularly preferably propyl, butyl, pentyl,    cyclopentyl or cyclohexyl, whereas propyl, pentyl, cyclopentyl or    cyclohexyl, particularly cyclopentyl and cyclohexyl are of    particular importance,    and the groups L, R¹, R², R⁴ and R⁵ may have one of the meanings    given above or hereinafter,    optionally in the form of the tautomers, racemates, enantiomers,    diastereomers and mixtures thereof, and optionally in the form of    the pharmacologically acceptable acid addition salts, solvates    and/or hydrates thereof.

Of particular importance according to the invention are compounds ofgeneral formula (I),

wherein

-   R⁴ denotes hydrogen, methyl, ethyl, methyloxy or ethyloxy,    preferably hydrogen, methyl or methyloxy, particularly preferably    hydrogen or methyloxy,    and the groups L, R¹, R², R³ and R⁵ may have one of the meanings    given above or hereinafter,    optionally in the form of the tautomers, racemates, enantiomers,    diastereomers and mixtures thereof, and optionally in the form of    the pharmacologically acceptable acid addition salts, solvates    and/or hydrates thereof.

Of particular importance according to the invention are compounds ofgeneral formula (I),

wherein

-   R⁵ denotes a group selected from among cyclopropyl, cyclopentyl or    cyclohexyl, which may optionally be mono- or disubstituted,    preferably monosubstituted, by methyl, ethyl, OH, fluorine,    chlorine, CF₃, piperidinyl, morpholinyl, pyrrolidinyl or    piperazinyl, while the above-mentioned possible substituents    piperidinyl, morpholinyl, pyrrolidinyl and piperazinyl may in turn    be mono- or disubstituted by methyl, ethyl, cyclopropyl or    cyclopropylmethyl,    and the groups L, R¹, R², R³ and R⁴ may have one of the meanings    given above or hereinafter,    optionally in the form of the tautomers, racemates, enantiomers,    diastereomers and mixtures thereof, and optionally in the form of    the pharmacologically acceptable acid addition salts, solvates    and/or hydrates thereof.

Of particular importance according to the invention are compounds ofgeneral formula (I),

wherein

-   R⁵ denotes a group selected from among cyclopentyl or cyclohexyl,    which may optionally be mono- or disubstituted, preferably    monosubstituted, by methyl, fluorine, CF₃, morpholinyl or    piperazinyl, while the above-mentioned possible substituents    morpholinyl and piperazinyl may in turn be mono- or disubstituted,    preferably monosubstituted, by methyl, ethyl or cyclopropylmethyl,    and the groups L, R¹, R², R³ and R⁴ may have one of the meanings    given above or hereinafter,    optionally in the form of the tautomers, racemates, enantiomers,    diastereomers and mixtures thereof, and optionally in the form of    the pharmacologically acceptable acid addition salts, solvates    and/or hydrates thereof.

Of particular importance according to the invention are compounds ofgeneral formula (I),

wherein

-   R⁵ denotes a heteroaryl selected from among imidazolyl, oxazolyl,    isoxazolyl, pyrolyl, pyrazolyl, oxadiazolyl, pyridyl, pyridazinyl,    pyrazinyl, pyrimidinyl, indolyl, quinolinyl, isoquinolinyl,    benzimidazolyl, purinyl and pteridinyl, which may optionally be    mono- or disubstituted, preferably monosubstituted, by fluorine,    chlorine, OH, CF₃, methyl, ethyl, propyl, phenyl, benzyl or    phenethyl,    and the groups L, R¹, R², R³ and R⁴ may have one of the meanings    given above or hereinafter,    optionally in the form of the tautomers, racemates, enantiomers,    diastereomers and mixtures thereof, and optionally in the form of    the pharmacologically acceptable acid addition salts, solvates    and/or hydrates thereof.

Of particular importance according to the invention are compounds ofgeneral formula (I),

wherein

-   R⁵ denotes a heteroaryl selected from among imidazolyl, pyrolyl,    pyridyl, pyrimidinyl, quinolinyl, isoquinolinyl and benzimidazolyl,    preferably selected from pyridyl, pyrollyl and pyrimidinyl, which    may optionally be mono- or disubstituted, preferably    monosubstituted, by fluorine, chlorine, CF₃, methyl, ethyl, phenyl    or benzyl,    and the groups L, R¹, R², R³ and R⁴ may have one of the meanings    given above or hereinafter,    optionally in the form of the tautomers, racemates, enantiomers,    diastereomers and mixtures thereof, and optionally in the form of    the pharmacologically acceptable acid addition salts, solvates    and/or hydrates thereof.

Of particular importance according to the invention are compounds ofgeneral formula (I),

wherein

-   R⁵ denotes a heteroaryl selected from among imidazolyl, pyrolyl,    pyridyl, pyrimidinyl, quinolinyl, isoquinolinyl and benzimidazolyl,    preferably selected from pyridyl, pyrolyl and pyrimidinyl, which may    optionally be mono- or disubstituted, preferably monosubstituted, by    fluorine, chlorine, CF₃, methyl, ethyl, phenyl or benzyl,    and the groups L, R¹, R², R³ and R⁴ may have one of the meanings    given above or hereinafter,    optionally in the form of the tautomers, racemates, enantiomers,    diastereomers and mixtures thereof, and optionally in the form of    the pharmacologically acceptable acid addition salts, solvates    and/or hydrates thereof.

The term alkyl groups, including alkyl groups which are a part of othergroups, denotes branched and unbranched alkyl groups with 1 to 8 carbonatoms, preferably 1-6, most preferably 1-4 carbon atoms. Examplesinclude: methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl and octyl.Unless otherwise stated, the abovementioned terms propyl, butyl, pentyl,hexyl, heptyl and octyl include all the possible isomeric forms. Forexample, the term propyl includes the two isomeric groups n-propyl andiso-propyl, the term butyl includes n-butyl, iso-butyl, sec. butyl andtert.-butyl, the term pentyl includes iso-pentyl, neopentyl, etc.

In the abovementioned alkyl groups, unless stated to the contrary, oneor more hydrogen atoms may optionally be replaced by other groups. Forexample these alkyl groups may be substituted fluorine. All the hydrogenatoms of the alkyl group may optionally also be replaced.

By alkyloxy groups, optionally also known as alkoxy groups or —O-alkylgroups, are meant the above-mentioned alkyl groups which are linked byan oxygen bridge. Examples include: methyloxy, ethyloxy, propyloxy,butyloxy, pentyloxy, hexyloxy, heptyloxy and octyloxy, which areoptionally also known as methoxy, ethoxy, propoxy etc.

By alkylene bridges or alkylene groups are meant, unless statedotherwise, branched and unbranched alkyl groups with 1 to 6 carbonatoms, for example methylene, ethylene, propylene, isopropylene,n-butylene, iso-butyl, sec. butyl and tert.-butyl etc. bridges.Particularly preferred are methylene, ethylene, propylene and butylenebridges. In the above-mentioned alkylene bridges, unless statedotherwise or additionally defined, 1 to 2 C atoms may optionally bereplaced by one or more heteroatoms selected from among oxygen, nitrogenor sulphur.

By alkenyl groups (including those which are a part of other groups) aremeant branched and unbranched alkylene groups with 2 to 8 carbon atoms,preferably 2-6 carbon atoms, particularly preferably 2-3 carbon atoms,provided that they have at least one double bond. Examples include:ethenyl, propenyl, butenyl, pentenyl, etc. Unless otherwise specified,the terms propenyl, butenyl etc. used above encompass all the possibleisomeric forms. For example the term butenyl includes 1-butenyl,2-butenyl, 3-butenyl, 1-methyl-1-propenyl, 1-methyl-2-propenyl,2-methyl-1-propenyl, 2-methyl-2-propenyl and 1-ethyl-1-ethenyl.

In the above-mentioned alkenyl groups, unless otherwise stated, one ormore hydrogen atoms may optionally be replaced by other groups. Forexample these alkyl groups may be substituted by halogen atoms in theform of fluorine. All the hydrogen atoms of the alkenyl group mayoptionally also be replaced.

Examples of alkenyloxy groups, optionally also known as alkenoxy groupsor —O-alkenyl groups, are the above-mentioned alkenyl groups which arelinked by an oxygen bridge. Examples include: ethylenoxy, propylenoxy,butylenoxy.

Examples of alkenylene groups (including those which are part of othergroups) include branched and unbranched, bridging alkylene groups with 2to 6 carbon atoms, preferably 2-4 carbon atoms, particularly preferably2-3 carbon atoms, provided that they have at least one double bond.Examples include: ethenylene, propenylene etc. Unless stated otherwise,the terms propenylene, butenylene etc. used above include all thepossible isomeric forms.

Examples of alkynyl groups (including those which are part of othergroups) are branched and unbranched alkynyl groups with 2 to 8 carbonatoms, provided that they have at least one triple bond, for exampleethynyl, propargyl, butynyl, pentynyl, hexynyl etc., preferably ethynylor propynyl.

In the above-mentioned alkynyl groups, unless otherwise stated, one ormore hydrogen atoms may optionally be replaced by other groups. Forexample these alkynyl groups may be substituted by fluorine. All thehydrogen atoms of the alkynyl group may optionally also be replaced.

Examples of alkynyloxy groups, optionally also known as alkynoxy groupsor —O-alkynyl groups, are the above-mentioned alkynyl groups which arelinked by an oxygen bridge.

Examples of alkynylene groups (including those which are part of othergroups) are branched and unbranched, bridging alkynyl groups with 2 to 6carbon atoms, provided that they have at least one triple bond, forexample ethynylene, propargylene etc. In the above-mentioned alkynylenegroups, unless otherwise stated, one or more hydrogen atoms mayoptionally be replaced by other groups. Unless stated otherwise, theterms propargylene etc. used above include all the possible isomericforms.

The term aryl denotes an aromatic ring system with 6 to 14 carbon atoms,preferably 6 or 10 carbon atoms, preferably phenyl or naphthyl,particularly preferably phenyl.

By 5-10-membered heteroaryl groups, which may contain one, two or threeheteroatoms selected from among nitrogen, oxygen and sulphur, preferablynitrogen or oxygen, are meant mono- or bicyclic aromatic ring systemswhich are selected, for example, from among imidazolyl, oxazolyl,isoxazolyl, pyrolyl, pyrazolyl, oxadiazol, pyridyl, pyridazinyl,pyrazinyl, pyrimidinyl, indolyl, quinolinyl, isoquinolinyl,benzimidazolyl, purinyl, pyrimidopyrimidinyl, benzoxazolyl,benzisoxazolyl, pyridopyrimidinyl and pteridinyl.

Examples of cycloalkyl groups are cycloalkyl groups with 3-8 carbonatoms, for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl or cyclooctyl, preferably cyclopropyl, cyclopentyl orcyclohexyl. A part from cyclopropyl and cyclobutyl, the above-mentionedcycloalkyl groups may optionally also be partially unsaturated, i.e.they may contain at least one double bond such as for examplecyclohexene. The term cylcoalkylene group denotes bridging,double-bonded cycloalkyl groups.

“═O” denotes an oxygen atom linked by a double bond.

Examples of 5-10-membered heterocycloalkyl groups which may contain one,two or three heteroatoms selected from among nitrogen, oxygen andsulphur, preferably nitrogen or oxygen, include, unless stated otherwisein the definitions, for example tetrahydrofuranyl, tetrahydrofuranonyl,γ-butyrolactonyl, α-pyranyl, γ-pyranyl, dioxolanyl, tetrahydropyranyl,dioxanyl, dihydrothiophenyl, thiolanyl, dithiolanyl, pyrrolinyl,pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, diazepanyl,oxazinyl, tetrahydrooxazinyl, pyrazolidinyl, preferably morpholinyl,pyrrolidinyl, piperidinyl and piperazinyl.

Halogen generally denotes fluorine, chlorine, bromine or iodine,preferably fluorine, chlorine or bromine, particularly preferablyfluorine or chlorine.

The compounds according to the invention may be present in the form ofthe individual optical isomers, mixtures of the individual enantiomers,diastereomers or racemates, in the form of the tautomers and in the formof the free bases or the corresponding acid addition salts withpharmacologically acceptable acids. By acid addition salts withpharmacologically acceptable acids are meant, for example, the saltsselected from among the hydrochloride, hydrobromide, hydroiodide,hydrosulphate, hydrophosphate, hydromethanesulphonate, hydronitrate,hydromaleate, hydroacetate, hydrobenzoate, hydrocitrate, hydrofumarate,hydrotartrate, hydrooxalate, hydrosuccinate, hydrobenzoate andhydro-p-toluenesulphonate, preferably the hydrochloride, hydrobromide,hydrosulphate, hydrophosphate, hydrofumarate and hydromethanesulphonate.

Of the above-mentioned acid addition salts the salts of hydrochloricacid, methanesulphonic acid, benzoic acid and acetic acid areparticularly preferred according to the invention.

Of the enantiomers and diastereomeric compounds of general formula (I)which may optionally exist, the optical isomers which have the Rconfiguration at the carbon centre carrying the two groups R¹ and R² arepreferred according to the invention

The group R⁴, if it is not hydrogen, may be linked in the ortho or metaposition in relation to the NH group linked to the pteridinone structurein the compounds of general formula (I). Particularly preferred arethose compounds of general formula (I) wherein R⁴ is in the orthoconfiguration relative to the above-mentioned NH group. These preferredcompounds are characterised by general formula (I′)

wherein the groups L, R¹, R², R³, R⁴ and R⁵ may have the above-mentionedmeanings, optionally in the form of the tautomers, racemates,enantiomers, diastereomers and mixtures thereof, and optionally in theform of the pharmacologically acceptable acid addition salts, solvatesand/or hydrates thereof.

The compounds according to the invention may be prepared by synthesismethod A described hereinafter, the substituents of general formulae(A1) to (A9) being defined as hereinbefore. This method is to beunderstood as illustrating the invention without restricting it to thecontent thereof.

Method A

Step 1A

A compound of formula (A1) is reacted with a compound of formula (A2) toproduce a compound of formula (A3) (Diagram 1A). This reaction may becarried out according to WO 0043369 or WO 0043372. Compound (A1) iscommercially obtainable, for example from City Chemical LLC, 139 AllingsCrossing Road, West Haven, Conn., 06516, USA. Compound (A2) may beprepared by methods described in the literature a) F. Effenberger, U.Burkhart, J. Willfahrt Liebigs Ann. Chem. 1986, 314-333. b) T. Fukuyama,C.-K. Jow, M. Cheung, Tetrahedron Lett. 1995, 36, 6373-6374. c) R. K.Olsen, J. Org. Chem. 1970, 35, 1912-1915. d) F. E. Dutton, B. H. ByungTetrahedron Lett. 1998, 30, 5313-5316. e) J. M. Ranajuhi, M. M. JoullieSynth. Commun. 1996, 26, 1379-1384.

In Step 1A, 1 equivalent of the compound (A1) and 1 to 1.5 equivalents,preferably 1.1 equivalents of a base, preferably potassium carbonate,potassium hydrogen carbonate, sodium carbonate, sodium hydrogencarbonate or calcium carbonate, particularly preferably potassiumcarbonate, are stirred in a diluent, optionally mixed with water, forexample acetone, tetrahydrofuran, diethyl ether, cyclohexane,methylcyclohexane, petroleum ether or dioxane, preferably cyclohexane ordiethyl ether.

At a temperature of 0 to 15° C., preferably 5 to 10° C., 1 equivalent ofan amino acid of formula (A2) dissolved in an organic solvent, forexample acetone, tetrahydrofuran, diethyl ether, cyclohexane or dioxane,is added dropwise. The reaction mixture is heated to a temperature of18° C. to 30° C., preferably about 22° C., with stirring and thenstirred for a further 10 to 24 hours, preferably about 12 hours. Thenthe diluent is distilled off, the residue is combined with water and themixture is extracted two to three times with an organic solvent, forexample, diethyl ether or ethyl acetate, preferably ethyl acetate. Thecombined organic extracts are dried and the solvent is distilled off.The residue (compound A3) may be used in Step 2 without any priorpurification.

Step 2A

The compound (A3) obtained in Step 1A is reduced at the nitro group andcyclised to form the compound of formula (A4) (Diagram 2A).

In Step 2A 1 equivalent of the nitro compound (A3) is dissolved in anacid, preferably glacial acetic acid, formic acid or aqueoushydrochloric acid, preferably glacial acetic acid, and heated to 50 to70° C., preferably about 60° C. Then a reducing agent, for example zinc,tin or iron, preferably iron powder, is added until the exothermicreaction has ended and the mixture is stirred for 0.2 to 2 hours,preferably 0.5 hours, at 100 to 125° C., preferably at about 115° C.After cooling to ambient temperature the iron salt is filtered off andthe solvent is distilled off. The residue is taken up in a solvent ormixture of solvents, for example ethyl acetate ordichloromethane/methanol 9/1 and semisaturated NaCl solution andfiltered through kieselguhr for example. The organic phase is dried andevaporated down. The residue (compound (A4)) may be purified bychromatography or by crystallisation or used as the crude product inStep 3A of the synthesis.

Step 3A

The compound (A4) obtained in Step 2A may be reacted by electrophilicsubstitution according to Diagram 3A to form the compound of formula(A5).

In Step 3A 1 equivalent of the amide of formula (A4) is dissolved in anorganic solvent, for example dimethylformamide or dimethylacetamide,preferably dimethylacetamide, and cooled to about −5 to 5° C.,preferably 0° C.

Then 0.9 to 1.3 equivalents sodium hydride and 0.9 to 1.3 equivalents ofa methylating reagent, for example methyliodide, are added. The reactionmixture is stirred for 0.1-3 hours, preferably about 1 hour, at about 0to 10° C., preferably at about 5° C., and may optionally be left tostand for a further 12 hours at this temperature range. The reactionmixture is poured onto ice water and the precipitate is isolated. Theresidue (compound (A5)) may be purified by chromatography, preferably onsilica gel, or by crystallisation or used as the crude product in Step4A of the synthesis.

Step 4A

The amination of the compound (A5) obtained in Step 3A to form thecompound of formula (I) (Diagram 4A) may be carried out according to themethods of variants 4.1A known from the literature from (a) M. P. V.Boarland, J. F. W. McOmie J. Chem. Soc. 1951, 1218-1221 or (b) F. H. S.Curd, F. C. Rose J. Chem. Soc. 1946, 343-348, or 4.2A from (a) Banks J.Am. Chem. Soc. 1944, 66, 1131, (b) Ghosh and Dolly J. Indian Chem. Soc.1981, 58, 512-513 or (c) N. P. Reddy and M. Tanaka Tetrahedron Lett.1997, 38, 4807-4810.

For example in variant 4.1A, 1 equivalent of the compound (A5) and 1 to3 equivalents, preferably about 1 equivalent of the compound (A6) may beheated without a solvent or with an organic solvent such as for examplesulpholane, dimethylformamide, dimethylacetamide, toluene,N-methylpyrrolidone, dimethylsulphoxide, or dioxane, preferablysulpholane over 0.1 to 4 hours, preferably 1 hour, at 100 to 220° C.,preferably at about 160° C. After cooling the product (A9) iscrystallised by the addition of org. solvents or mixtures of solvents,e.g. diethyl ether/methanol, ethyl acetate, methylene chloride, ordiethyl ether, preferably diethyl ether/methanol 9/1, or purified bychromatography.

As can be seen from Diagram 4A, the compounds of formula (A5) are ofcentral importance for the synthesis of the compounds of general formula(I) according to the invention. Accordingly, the present invention alsorelates to intermediate compounds of general formula (A5)

wherein the groups R¹, R² and R³ may have the above-mentioned meanings,optionally in the form of the tautomers, racemates, enantiomers,diastereomers and mixtures thereof, and optionally in the form of theacid addition salts, solvates and/or hydrates thereof.

For example in variant 4.2A, 1 equivalent of the compound (A5) and 1 to3 equivalents of the compound (A6) are refluxed for 1 to 48 hours,preferably about 5 hours, with acid, for example 1-10 equivalents of10-38% hydrochloric acid and/or an alcohol such as ethanol, propanol,dioxane or butanol, preferably ethanol, with stirring.

The precipitated product of formula (I) is filtered off and optionallywashed with water, dried and crystallised from a suitable org. solvent.

The products of formula (A7) are obtained analogously to the processesdescribed and are converted into the products of formula (I) by furtherreactions.

Step 5A

After the amination in Step 4A the products of formula (A9) may also beobtained by cleaving an acid- or base-labile group, for example, fromcompounds of type (A7) or by reduction of a nitro group to the amine(A8) and then reacting it to form amides (A9a), urethanes (A9b) or ureas(A9c) (cf. Diagram 5A).

Variant 5.1A:

For example, 1 equivalent of a compound (A7a) is combined with anacid-labile protective group, for example tert-butyloxycarbonyl with1-50 equivalents of acid, preferably HCl or trifluoroacetic acid, in anorganic solvent e.g. methylene chloride, ether, dioxane ortetrahydrofuran, preferably methylene chloride and stirred for 1 to 24 hat 20-100° C., preferably 20° C. The reaction mixture is separated forexample on silica gel or obtained by suitable crystallisation.

Variant 5.2B:

For example, 1 equivalent of a compound (A7b) is dissolved in a solvente.g. methanol, ethanol, THF, ethyl acetate or water and combined with0.001 to 0.1 equivalent Pd/C (10%) and hydrogenated with hydrogen for1-24 h. After filtration of the catalyst the product (A8) is obtainedand is optionally purified by silica gel chromatography or by suitablecrystallisation.

The group PG, as shown in the above diagram at compounds (A7a), may beone of the amino protecting groups known in the art. Suitable methods ofcleaving the group PG and hence converting the compounds into thecompounds of formula (A8) are known in the art (cf. T. W. Greene,“Protective Groups in Organic Synthesis”, 2nd Edition).

The compounds of formulae (A9a), (A9b) and (A9c) shown in Diagram 5A arespecific examples of the compounds of formula (I) according to theinvention. In the compounds of formula (A9a) L does not represent agroup L which is linked to the carbonyl carbon by an —NH or —O— bridge.Rather, these compounds are represented by formulae (A9b) and (A9c).

Preparation of the Amides (A9a)

For example, 1 equivalent of the compound (A8) is dissolved with 1equivalent of an activating reagent, e.g.O-benzotriazolyl-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU)and a base, for example about 1.5 equivalents, diisopropylethylamine(DIPEA) in an organic diluent, for example dichloromethane,tetrahydrofuran, dimethylformamide, N-methylpyrrolidone ordimethylacetamide, preferably dichloromethane or dimethylformamide.After the addition of 1 equivalent of the amine (A 10) the reactionmixture is stirred for 0.1 to 24 hours, preferably about 2 hours at 20°C. to 100° C. The product of formula (A9a) is obtained for example bycrystallisation or chromatographic purification.

Preparation of the Ureas (A9b)

The compounds A9b mentioned in Diagram 5A are compounds wherein Ldenotes an NH group. The processes described hereinafter may also beused if L represents not only NH but also —NH-alkylene, for example, aswill be apparent to the skilled man.

Variant A:

1 equivalent of amine (A8) is dissolved in an organic solvent, forexample dichloromethane, THF or dimethylformamide, and a base, forexample pyridine, triethylamine or disopropylethylamine, and combinedwith 1-2 equivalents, preferably 1 equivalent, of 4-nitrophenylchloroformate. After 1-24 h, preferably 2-5 h, 1 equivalentH₂N-L_(n)-R⁵, dissolved in an organic solvent, is added and the mixtureis stirred for 4-24 h at 20° C. The product of formula (A9b) is obtainedfor example by crystallisation or chromatographic purification.

Variant B:

1 equivalent of the amine (A8) is dissolved together with 1-3equivalents of an isocyanate in an organic solvent such asdimethylformamide, THF or dimethylacetamide and stirred for 1-24 h at40-70° C.

After the solvent has been eliminated the product (A9b) is obtained forexample by crystallisation or chromatographic purification.

Preparation of the Urethanes (A9c)

The compounds A9c shown in Diagram 5A are compounds wherein L denotes an—O— group. The processes described hereinafter may also be used if Lrepresents not only O but also —O-alkylene, for example, as will beapparent to the skilled man.

1 equivalent of the amine (A7) is dissolved in an organic solvent, suchas dichloromethane, dimethylformamide or THF and combined with 1-3equivalents of base, for example diisopropylethylamine or triethylamine.Subsequently 1-3 equivalents of a chloroformate are added and themixture is stirred for 1-24 h at 20-60° C. After the solvent has beeneliminated the product (A9c) is obtained for example by crystallisationor chromatographic purification.

As can be seen from Diagram 5A, the compounds of formula (A8) are ofcentral importance in the synthesis of the compounds of general formula(I) according to the invention. Accordingly, the present invention alsorelates to intermediate compounds of general formula (A8)

wherein the groups R¹, R², R³ and R⁴ may have the above-mentionedmeanings, optionally in the form of the tautomers, racemates,enantiomers, diastereomers and mixtures thereof, and optionally in theform of the acid addition salts, solvates and/or hydrates thereof.

Examples of acid addition salts which may be used particularly includethose salts mentioned hereinbefore for the compounds of formula (I) asbeing pharmacologically acceptable acid addition salts.

As can be seen from Diagram 5A, the compounds of formula (A7a) are alsoof major importance in the synthesis of the compounds of general formula(I) according to the invention. Accordingly, the present invention alsorelates to intermediate compounds of general formula (A7a)

wherein the groups R¹, R², R³ and R⁴ may have the meanings given aboveand wherein PG denotes an amino protecting group, optionally in the formof the tautomers, racemates, enantiomers, diastereomers and mixturesthereof, and optionally in the form of the acid addition salts, solvatesand/or hydrates thereof.

Preferred are compounds of general formula (A7a), wherein

-   PG is selected from among tert-butyloxycarbonyl, acetyl,    trifluoromethyl, 9-fluoroenylmethyloxycarbonyl, allyloxycarbonyl and    benzyloxycarbonyl, preferably tert-butyloxycarbonyl, acetyl and    trifluoromethyl,    optionally in the form of the tautomers, racemates, enantiomers,    diastereomers and mixtures thereof, and optionally in the form of    the acid addition salts, solvates and/or hydrates thereof.

As can be seen from Diagram 5A, the compounds of formula (A7b) are alsoof major importance in the synthesis of the compounds of general formula(I) according to the invention. Accordingly, the present invention alsorelates to intermediate compounds of general formula (A7b)

wherein the groups R¹, R², R³ and R⁴ may have the meanings given above,optionally in the form of the tautomers, racemates, enantiomers,diastereomers and mixtures thereof, andoptionally in the form of the acid addition salts, solvates and/orhydrates thereof.

The preparation of a reactant used to synthesise specific intermediatesof general formula (A8), the intermediate compounds Z1-Z7, is describedbelow.

Preparation of tert-butyl 4-amino-3-methoxy-phenyl-carbamate

15 g of 4-nitro-3-methoxybenzoic acid was dissolved in 35 gtert-butanol, 16 mL disopropylethylamine and 15 mL toluene, thencombined with 17.5 mL diphenylphosphorylazide and refluxed for 7 h. Themixture was then evaporated down and combined with 500 mL ethyl acetateand extracted with 3×200 mL water. The org. phase was dried and thereaction mixture was separated by silica gel chromatography (petroleumether:ethyl acetate 3:1), and suitable fractions were combined.

Yield: 16.1 g of a compound B1 (bright yellow crystals)

16 g of the compound B1 was dissolved in 400 mL ethanol and reacted with6 g 10% Pd/C with hydrogen at 20° C. The reaction solution wasevaporated down and the solid was triturated with diethyl ether.

Yield: 10.5 g tert-butyl 4-amino-3-methoxy-phenyl-carbamate (colourlesscrystals)

In order to synthesise Examples 1 to 3 first of all an intermediatecompound Z1 is prepared as described below.

50.0 g D-alanine methylester x HCl and 49.1 g cyclohexanone were placedin 300 mL dichloromethane and then combined with 41.0 g sodium acetateand 159.0 g sodium triacetoxyborohydride. The mixture was stirredovernight and then 300 mL of 10% sodium hydrogen carbonate solution wereadded. The aqueous phase was extracted with dichloromethane. Thecombined organic phases were washed with 10% sodium hydrogen carbonatesolution, dried over Na₂SO₄ and evaporated down.

Yield: 72.5 g of a compound Z1a (clear liquid)

72.5 g of the compound Z1a were placed in 500 mL water and 76.6 g (0.39mol) 2,4-dichloro-5-nitropyrimidine in 500 mL diethyl ether were added.At a temperature of −5° C. 100 mL 10% potassium hydrogen carbonatesolution were added dropwise. The mixture was stirred for 3 h at −5° C.and for a further 12 h at ambient temperature. The organic phase wasseparated off and dried over Na₂SO₄. During evaporation the productcrystallises out.

Yield: 48.0 g of a compound Z1b (yellow crystals)

48.0 g of the compound Z1b were dissolved in 350 mL glacial acetic acidand heated to 60° C. 47.5 g iron powder were added batchwise, while thetemperature rose to 105° C. The reaction mixture was stirred for threehours at 80° C., then filtered hot through cellulose and evaporateddown. The residue was stirred in water and ethyl acetate, suctionfiltered and the light grey precipitate was washed with ethyl acetate.The filtrate was washed with dilute ammonia and water, the organic phasewas dried over Na₂SO₄, filtered through activated charcoal andevaporated down.

Yield: 29.5 g of a compound Z1c (light grey crystals)

32.1 g of the compound Z1c were placed in 300 mL dimethylacetamide andcombined with 13 mL (0.2 mol) methyliodide. At −5° C. 6.4 g (0.16 mol)sodium hydride were added batchwise as a 60% dispersion in mineral oil.After 2 h the reaction mixture was poured onto 800 mL ice water. Theprecipitate formed was suction filtered and washed with petroleum ether.

Yield: 33.0 g of a compound Z1d (beige crystals)

8 g of Z1d were refluxed for 12 h together with 5.54 g4-trifluoracetyl-p-phenylenediamine in a solution of 40 mL ethanol, 40mL water and 10 mL hydrochloric acid. The ethanol was then eliminated invacuo, the reaction mixture was cooled and the precipitate formed wasfiltered off. The mother liquor was evaporated down and the residue wastriturated with ethanol.

Yield: 5.86 g of the intermediate compound Z1 as a beige solid

In order to synthesise Examples 17 to 20 first of all an intermediatecompound Z2 is prepared as described below.

A solution of 128.2 g (0.83 mol) D-alanine ethylester x HCl and 71.5 g(0.85 mol) cyclopentanone in 1500 mL dichloromethane was combined with70.1 (0.85 mol) sodium acetate and 265.6 g (1.25 mol) sodiumtriacetoxyborohydride. The reaction mixture was stirred for 12 h andthen poured into 1.5 L of a 10% sodium hydrogen carbonate solution. Theaqueous phase was extracted with dichloromethane. The combined organicphases were dried over Na₂SO₄ and evaporated down.

Yield: 143.4 g of a compound Z2a (colourless oil)

66.0 g of the compound Z2a were placed in 500 mL water and combined with85.0 g (0.44 mol) 2,4-dichloro-5-nitropyrimidine in 500 mL diethylether. At −5° C. 100 mL 10% potassium hydrogen carbonate solution wereadded dropwise and the reaction mixture was stirred for 48 h at ambienttemperature. The aqueous phase was extracted with diethyl ether, thecombined organic phases were dried over Na₂SO₄ and evaporated down. Thedark red solid was extracted with petroleum ether and suction filtered.

Yield: 88.0 g of a compound Z2b (yellow crystals)

88.0 g of the compound Z2b were dissolved in 1000 mL glacial acetic acidand at 60° C. 85 g iron powder were added batchwise, while thetemperature rose to 110° C. The mixture was stirred for 1 h at 60° C.,then suction filtered hot through cellulose and evaporated down. Thebrown solid was stirred with 700 mL water and suction filtered.

Yield: 53.3 g of a compound Z2c (light brown crystals)

53.3 g of the compound Z2c were dissolved in 300 mL dimethylacetamideand combined with 13 mL (0.21 mol) methyl iodide. At −5° C. 5.0 g (0.21mol) sodium hydride were added batchwise as 60% dispersion in mineraloil. After 12 h the reaction mixture was poured onto 1000 mL ice waterand the precipitate formed was suction filtered.

Yield: 40.0 g of a compound Z2d (colourless crystals)

1.95 g of Z2d and 1.66 g tert-butyl 4-amino-3-methoxy-phenyl-carbamatewere melted together at 120° C. for 4.5 h. After cooling the reactionmixture was dissolved in dichloromethane and extracted 1× with potassiumcarbonate solution and 2× with water. After drying the org. phase themixture was separated by silica gel chromatography (eluant 99:1,CH₂Cl₂:MeOH) and the product fractions were combined.

Yield: 1.76 g of the compound Z2e (brown solid)

1.75 g of Z2e was dissolved in 100 mL methylene chloride and thesolution was combined with 20 mL trifluoroacetic acid. After 12 hstirring at 25° C. the reaction mixture was added to semiconcentratedammonia solution while being cooled and the org. phase was separated offand extracted with water. After elimination of the solvent the mixturewas dissolved in acetone and combined with ethereal HCl. The precipitateformed was filtered off and dried.

Yield: 1.32 g of the intermediate compound Z2

In order to synthesise Examples 13 and 21 to 23 first of all anintermediate compound Z3 is prepared as described below.

54.0 g (0.52 mol) D-2-aminobutyric acid were suspended in 540 mLmethanol and 132 g (1.1 mol) thionyl chloride were slowly added whilecooling with ice. The mixture was refluxed for 1.5 h and then evaporateddown. The oil remaining was combined with 540 mL tert-butylmethyletherand the colourless crystals obtained were suction filtered.

Yield: 78.8 g of a compound Z3a (colourless crystals)

74.2 g of the compound Z3a and 43.5 mL (0.49 mol) cyclopentanone weredissolved in 800 mL dichloromethane. After the addition of 40.0 g (0.49mol) sodium acetate and 150.0 g (0.71 mol) sodium triacetoxyborohydrideat 0° C. the mixture was stirred for 12 h at ambient temperature andthen 500 mL 20% sodium hydrogen carbonate solution were added. Theaqueous phase was extracted with dichloromethane. The combined organicphases were washed with water, dried over MgSO₄ and evaporated down.

Yield: 85.8 g of a compound Z3b (light yellow oil)

40.0 g of the compound Z3b and 30.0 g (0.22 mol) potassium carbonatewere suspended in 600 mL acetone and while cooling with ice combinedwith 45.0 g (0.23 mol) 2,4-dichloro-5-nitropyrimidine in 200 mL acetone.After 12 h a further 5.0 g of 2,4-dichloro-5-nitropyrimidine were addedand the mixture was stirred for 3 h. The reaction mixture was evaporateddown, taken up in 800 mL ethyl acetate and 600 mL water and the aqueousphase was extracted with ethyl acetate. The combined organic phases werewashed with water, dried over MgSO₄ and evaporated down.

Yield: 75.0 g of a compound Z3c (brown oil)

100 g of the compound Z3c were dissolved in 650 mL glacial acetic acidand at 70° C. 20 g iron powder were added batchwise. The mixture wasstirred for 1 h at 70° C., then for 1.5 h at 100° C. and then filteredhot through kieselguhr. The reaction mixture was evaporated down, takenup in methanol/dichloromethane, applied to silica gel and purified bySoxhlet extraction with ethyl acetate. The solvent was removed and theresidue was stirred with methanol.

Yield: 30.0 g of a compound Z3d (light brown crystals)

25.0 g of the compound Z3d and 6.5 mL (0.1 mol) methyl iodide wereplaced in 250 mL dimethylacetamide and at −10° C. 3.8 g (0.95 mol)sodium hydride were added as a 60% dispersion in mineral oil. Themixture was stirred for 20 min. at 0° C., then 30 min. at ambienttemperature and finally ice was added. The reaction mixture wasevaporated down and combined with 300 mL water. The precipitate formedwas suction filtered and washed with petroleum ether.

Yield: 23.0 g of a compound Z3e (colourless solid)

1.5 g Z3e and 1.22 g tert-butyl 4-amino-3-methoxy-phenyl-carbamate weremelted together at 120° C. for 5 h. After cooling the reaction mixturewas dissolved in dichloromethane and extracted 2× with potassiumcarbonate solution and 2× with water. After drying the org. phase themixture was separated by silica gel chromatography (eluant 99:1,CH₂Cl₂:MeOH) and the product fractions were combined.

Yield: 0.92 g of a compound Z3f (light brown crystals)

0.92 g Z3f were dissolved in 100 mL methylene chloride, 15 mLtrifluoroacetic acid were added and the mixture was stirred for 3 h at20° C. Then the solution was added to a mixture of 10 g ice and 100 mLof a 25% ammonia solution and the org. phase was extracted with waterand evaporated down after drying. The residue was dissolved in acetoneand combined with ethereal HCl. The crystals precipitated were filteredoff and dried.

Yield: 0.54 g of the intermediate compound Z3 (light brown crystals)

In order to synthesise Examples 7 to 9 and 15 first of all anintermediate compound Z4 is prepared as described below.

A mixture of 73.4 mL ethyl 2-bromoisobutyrate, 87.1 mL3-methyl-1-butylamine, 82.5 g (0.6 mol) sodium iodide and 76.0 g (0.6mol) potassium carbonate in 1000 mL ethyl acetate was refluxed for 3days. Any salts present were filtered off and the filtrate wasevaporated down.

Yield: 97.0 g of a compound Z4a (red oil)

49.0 g 2,4-dichloro-5-nitropyrimidine and 38.3 g potassium carbonatewere suspended in 500 mL acetone and at 0° C. combined with 93.0 g ofthe compound Z4a in 375 mL acetone. The reaction mixture was stirredovernight at ambient temperature, filtered and evaporated down. Theresidue dissolved in ethyl acetate was washed with water and the organicphase was dried over MgSO₄ and evaporated down.

Yield: 102.7 g of a compound Z4b (brown oil)

22.7 g of the compound Z4b were dissolved in 350 mL glacial acetic acidand at 60° C. 17.4 g of iron powder were added batchwise. After theaddition had ended the mixture was refluxed for 0.5 h, filtered hot andevaporated down. The residue was taken up in 200 mLdichloromethane/methanol (9:1) and washed with sodium chloride solution.The organic phase was suction filtered through kieselguhr, dried overMgSO₄, evaporated down and separated by column chromatography (eluant:ethyl acetate/cyclohexane 1:1) and suitable fractions were combined.

Yield: 1.9 g of a compound Z4c (colourless crystals)

1.9 g of the compound Z4c were dissolved in 32 mL dimethylacetamide andwhile cooling with ice combined with 0.3 g (7 mmol) of sodium hydride asa 60% dispersion in mineral oil. After 10 min. 0.5 mL (7 mmol) of methyliodide were added and the mixture was stirred for 3 h at ambienttemperature. The reaction mixture was evaporated down and combined withwater. The precipitate formed was suction filtered and washed withpetroleum ether.

Yield: 1.6 g of a compound Z4d (colourless crystals)

1.5 g of Z4d and 1.21 g tert-butyl 4-amino-3-methoxy-phenyl-carbamatewere melted together at 120° C. for 4.5 h. After cooling the reactionmixture was dissolved in dichloromethane and extracted 1× with potassiumcarbonate solution and 1× with water. After drying the org. phase themixture was separated by silica gel chromatography (eluant 98:2,CH₂Cl₂:MeOH) and the product fractions were combined.

Yield: 1.12 g of a compound Z4e (light brown solid)

1.12 g Z4e was dissolved in 100 mL methylene chloride, 18 mLtrifluoroacetic acid were added and the mixture was stirred for 12 h at20° C. Then the solution was added to a semiconc. ammonia solution andthe org. phase was extracted with water and evaporated down.

Yield: 0.84 g of the intermediate compound Z4 (beige solid)

The following intermediates were prepared analogously to the synthesesdescribed above:

The preparation of some reactants used to synthesise the products isdescribed hereinafter.

The following acid may be obtained for example by the following methodknown from the literature:

1-methyl-piperidine-4-carboxylic acid

Gray, Allan P.; Platz, Robert D.; Henderson, Theresa R.; Chang, TimothyC. P.; Takahashi, Kazuyuki; Dretchen, Kenneth L, Journal of MedicinalChemistry (1988), 31(4), 807-14.

4-morpholinyl-4-yl-cyclohexanecarboxylic acid chloride

450 mg of methyl 4-amino-cyclohexanecarboxylate*hydrochloride (e.g.obtained according to the following literature: Johnston, Thomas P.;McCaleb, George S.; Clayton, Sarah D.; Frye, Jerry L.; Krauth, CharlesA.; Montgomery, John A. Journal of Medicinal Chemistry (1977), 20(2),279-90), 0.345 mL bis-(2-chloroethyl)ether and 1.4 g potassium carbonatewere dissolved in 4 mL dimethylformamide and heated together with 50 mgpotassium iodide to 100° C. for 3 h. Then 30 mL water were added and themixture was neutralised with acetic acid. The aqueous phase wasextracted with dichloromethane. After evaporation the mixture wasseparated by silica gel chromatography (methylene chloride:MeOH 15:1)and the suitable fractions were combined.

Yield: 259 mg of a product Z8a

233.3 mg Z8a was dissolved in 5 mL 1M sodium hydroxide solution and 4 mLmethanol and stirred for 12 h at 20° C. After elimination of themethanol the mixture was neutralised with 1M hydrochloric acid,evaporated down completely, the mixture was suspended in methanol andthe product was filtered off. It was then suspended in ethanol again,the insoluble residue was filtered off and the mother liquor wasevaporated down.

Yield: 206 mg of the acid Z8b (white solid)

206 mg of Z8b was refluxed in 150 μL thionyl chloride and 5 mL toluenefor 3 h. The mixture was then evaporated to dryness, the precipitate wasstirred overnight with ether and filtered off.

Yield: 204 mg of the intermediate Z8

The compound Z8 is not yet known in the art. In view of its importanceas a starting material for numerous compounds of formula (I) accordingto the invention, the present invention further relates to the compoundof formula Z8

trans-4-(4-cyclopropylmethyl-piperazin-1-yl)-cyclohexylamine

9.8 g N,N-dibenzyl-4-amino-cyclohexanone was dissolved in 100 mLdichloromethane and stirred with 5.6 g N-cyclopropylmethylpiperazine and8.5 g NaBH(OAc)₃ for 12 h at RT. Then the mixture was combined withwater and potassium carbonate, the org. phase was separated off, driedand the solvent was eliminated in vacuo. The residue was separated offusing a silica gel column (approx. 50 mL silica gel, approx. 3 L ethylacetate 95/methanol 5+0.25% conc. ammonia). The suitable fractions wereevaporated down in vacuo. The desired compound was crystallised fromethanol+conc. HCl.

Yield: 8.5 g of the compound Z9a.

8.5 g of Z14a were dissolved in 170 mL MeOH and hydrogenated on 1.7 gPd/C (10%) at 30-50° C. The solvent was eliminated in vacuo and theresidue was crystallised from ethanol and conc. HCl.

Yield: 4.4 g of the intermediate Z9.

trans-4-(2,6-dimethyl-morpholin-4-yl)-cyclohexylamine

110 mL benzylamine and 156 g cyclohexanedione monoethylene ketal wereheated in 800 mL toluene using a water separator. Then the solution wasevaporated down and the reaction mixture was taken up in 1000 mL ethanoland combined batchwise with a total of 23 g sodium borohydride whilebeing cooled to 15° C. After 12 h the reaction solution was evaporateddown and the residue was combined with 500 mL water, extracted 2× with400 mL ether, then the org. phase was washed with water, dried,evaporated down and the residue was distilled under a high vacuum.

Yield: 208 g of a product Z10a

208 g Z10a were heated together with 114 g benzyl chloride, 138 gpotassium carbonate and 14 g potassium iodide in 400 mLN-methylpyrrolidone for 24 h at 80° C. Subsequently 5 L water was addedand the solid formed was filtered off and washed with water again. Thenit was dissolved in 1 L methylene chloride, the org. phase was dried andevaporated down. The residue was crystallised from methanol.

Yield: 260 g of a product Z10b

260 g Z10b was dissolved in 400 mL water and 100 mL 37% HCl and stirredfor 4 h at 100°. After cooling the mixture was made alkaline withpotassium carbonate and the crystals precipitated were filtered off andwashed with water. The solid was dissolved in dichloromethane, the org.phase was dried and evaporated down. The residue was recrystallised frompetroleum ether.

Yield: 216 g of a product Z10c.

44 g Z10c and 23 g cis-2,6-dimethylmorpholine were refluxed in 100 mLtoluene and 0.1 mL methanesulphonic acid for 2 h using a waterseparator. The mixture was then cooled, the solvent was eliminated, and400 mL ethanol were added and 8 g sodium borohydride were addedbatchwise. The reaction temperature rose to 45° C., then after theexothermic reaction had died down the mixture was heated to 60° C. for 3h, cooled and combined with 400 mL water and, while being cooled,combined with 300 mL 2N HCl. Subsequently ethanol was added and themixture was combined with 400 mL 2N NaOH. The residue was extracted with2×300 mL dichloromethane. The org. phase was dried, evaporated down andcombined with 100 mL methanol, the solid was filtered off and the motherliquor was evaporated down. The residue was separated by silica gelchromatography and suitable fractions were combined.

Yield: 35.2 of a product Z10d

35 g Z10d was dissolved in 400 mL methanol and hydrogenated with 7 gPd/C (10%) at 50 psi hydrogen and 50° C. for 4 h. Subsequently themixture was evaporated down, suspended in 200 mL ethanol, 10 mL 37%hydrochloric acid were added dropwise, the mixture was stirred for 30minutes in the ice bath and the crystals were filtered off and washedwith cold ether and ethanol.

Yield: 24 g of the intermediate product Z10

The new compounds of general formula (I) may be synthesised analogouslyto the following synthesis examples. These Examples are however intendedonly as possible methods to illustrate the invention more fully withoutlimiting it to their content.

SYNTHESIS OF THE EXAMPLES Example 1

0.1 g Z1, 82 mg nicotinic acid chloride and 0.2 mL triethylamine werestirred for 1 h in 10 mL dichloromethane at 20° C., then the mixture wasextracted with water and the dried org. phase was evaporated down. Theresidue was crystallised from methanol and filtered off.

Yield: 0.025 g of a white solid

Example 7

0.1 g Z4, 23 μL cyclopropylcarboxylic acid chloride and 0.15 mLtriethylamine were stirred for 2 h in 2 mL dichloromethane at 20° C.,then the org. phase was extracted with 20 mL 5% aqueous potassiumcarbonate solution. The org. phase was evaporated down and the mixturewas separated by silica gel chromatography. The suitable fractions werecombined, evaporated down and the residue was crystallised from ethylacetate and petroleum ether.

Yield: 0.071 g white crystals

Example 16

0.1 g Z6, 65 mg 1-benzyl-piperidine-4-carboxylic acid chloride and 0.2mL triethylamine were stirred for 2 h in 2 mL dichloromethane at 20° C.,then the org. phase was extracted with 20 mL 5% aqueous potassiumcarbonate solution. The org. phase was evaporated down and the mixturewas separated by silica gel chromatography. The suitable fractions werecombined, evaporated down and the residue was crystallised from ethylacetate and petroleum ether.

Yield: 0.067 g white crystals

Example 23

0.1 g Z3, 76 mg 1-methyl-piperidine-4-carboxylic acid chloride and 0.3mL triethylamine were stirred for 2 h in 2 mL dichloromethane at 20° C.,then the org. phase was extracted with 20 mL 5% aqueous potassiumcarbonate solution. The org. phase was evaporated down and the mixturewas separated by silica gel chromatography. The suitable fractions werecombined, evaporated down and the residue was crystallised from ethylacetate and petroleum ether.

Yield: 0.064 g white crystals

Example 27

0.2 g Z5 is dissolved in 2 mL dichloromethane, 0.5 mL THF and 0.5 mLpyridine, then 144 mg 4-nitrophenyl chloroformate, dissolved in 1 mLdichloromethane, was added. After 3 h, 111 mg of1-methyl-piperidin-4-ylamine dissolved in 0.5 mL dichloromethane wereadded and the mixture was stirred for 12 h at 20° C. Thendichloromethane was added and the mixture was extracted 3× with water.The dried org. phase is evaporated down and the mixture is separated bysilica gel chromatography. Suitable fractions were combined, evaporateddown and crystallised from ethyl acetate and petroleum ether.

Yield: 97 mg as a grey solid

The compounds of formula (I) listed in Table 1, inter alia, are obtainedanalogously to the methods described hereinbefore.

The present invention relates, in particularly preferred embodiments, tothe compounds of formula (I), as listed in Table 1, per se, optionallyin the form of the tautomers, racemates, enantiomers, diastereomers andmixtures thereof, and optionally in the form of the pharmacologicallyacceptable acid addition salts, solvates and/or hydrates thereof.

TABLE 1 (I′)

Config. m.p. Example —C*(R¹R²)— —R¹ —R² —R³ —R⁴ —L— —R⁵ [° C.] 1 R —H—Me

—H —

276 (decomp.) 2 R —H —Me

—H —

219 3 R —H —Me

—H —

211 4 R —H —Me

—OMe —

178 5 R —H —Me

—OMe —

171 6 R —H —Me

—OMe —

 88 7 — —Me —Me

—OMe —

148 8 — —Me —Me

—OMe —

152 9 — —Me —Me

—OMe —

205 10 R —H —Me

—OMe —

157 11 R —H —Me

—OMe —

156 12 R —H —Me

—OMe —

161 13 R —H —Et

—OMe —

120 14 R —H —Me

—OMe —

180 15 — —Me —Me

—OMe —

180 16 R —H —Me

—OMe —

136 17 R —H —Me

—OMe —

135 18 R —H —Me

—OMe —

230 19 R —H —Me

—OMe —

197 20 R —H —Me

—OMe —

204 21 R —H —Et

—OMe —

136 22 R —H —Et

—OMe —

170 23 R —H —Et

—OMe —

112 24 R —H —Et

—OMe —

112 25 R —H —Et

—OMe —

134 26 R —H —Et

—OMe —

157 27 R —H —Et

—OMe —NH—

158 28 R —H —Et

—OMe —NH—

142 29 R —H —Et

—OMe —NH—

148 20 R —H —Et

—OMe —NH—

164

As has been found, the compounds of general formula (I) arecharacterised by their many possible applications in the therapeuticfield. Particular mention should be made of those applications for whichthe inhibition of specific cell cycle kinases, particularly theirinhibiting effect on the proliferation of cultivated human tumour cells,and also on the proliferation of other cells, such as e.g. endothelialcells, plays a part.

As demonstrated by DNA staining followed by FACS analysis, theinhibition of proliferation brought about by the compounds according tothe invention is mediated by the arrest of the cells above all in theG2/M phase of the cell cycle. The cells arrest, depending on the cellsused, for a specific length of time in this cell cycle phase beforeprogrammed cell death is initiated. An arrest in the G2/M phase of thecell cycle is initiated e.g. by the inhibition of specific cell cyclekinases. On the basis of their biological properties the compounds ofgeneral formula I according to the invention, their isomers and thephysiologically acceptable salts thereof are suitable for treatingdiseases characterised by excessive or anomalous cell proliferation.

Such diseases include for example: viral infections (e.g. HIV andKaposi's sarcoma); inflammatory and autoimmune diseases (e.g. colitis,arthritis, Alzheimer's disease, glomerulonephritis and wound healing);bacterial, fungal and/or parasitic infections; leukaemias, lymphomas andsolid tumours; skin diseases (e.g. psoriasis); bone diseases and;cardiovascular diseases (e.g. restenosis and hypertrophy). They are alsouseful for protecting proliferating cells (e.g. hair, intestinal, bloodand progenitor cells) from DNA damage caused by radiation, UV treatmentand/or cytostatic treatment (Davis et al., 2001). The new compounds maybe used for the prevention, short- or long-term treatment of theabove-mentioned diseases, also in combination with other activesubstances used for the same indications, e.g. cytostatics, hormones orantibodies.

The activity of the compounds according to the invention was determinedin the PLK1 inhibition assay, in the cytotoxicity test on cultivatedhuman tumour cells and/or in a FACS analysis, e.g. on HeLa S3 cells. Inboth test methods the compounds exhibited a good to very good activity,i.e. for example an EC₅₀ value in the HeLa S3 cytotoxicity test of lessthan 5 μmol/L, generally less than 1 μmol/L, and an IC₅₀ value in thePLK1 inhibition assay of less than 1 μmol/L.

PLK-1 Kinase Assay

Enzyme Preparation:

Recombinant human PLK1 enzyme linked to GST at its N-terminal end isisolated from insect cells infected with baculovirus (Sf21).Purification is carried out by affinity chromatography on glutathionesepharose columns.

4×10⁷ Sf21 cells (Spodoptera frugiperda) in 200 ml of Sf-900 II Serumfree insect cell medium (Life Technologies) are seeded in a spinnerflask. After 72 hours' incubation at 27° C. and 70 rpm, 1×10⁸ Sf21 cellsare seeded in a total of 180 ml medium in a new spinner flask. Afteranother 24 hours, 20 ml of recombinant Baculovirus stock suspension areadded and the cells are cultivated for 72 hours at 27° C. at 70 rpm. 3hours before harvesting, okadaic acid is added (Calbiochem, finalconcentration 0.1 μM) and the suspension is incubated further. The cellnumber is determined, the cells are removed by centrifuging (5 minutes,4° C., 800 rpm) and washed 1× with PBS (8 g NaCl/l, 0.2 g KCl/l, 1.44 gNa₂HPO₄/l, 0.24 g KH₂PO4/l). After centrifuging again the pellet isflash-frozen in liquid nitrogen. Then the pellet is quickly thawed andresuspended in ice-cold lysis buffer (50 mM HEPES pH 7.5, 10 mM MgCl₂, 1mM DTT, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 100 μM NaF, 100 μM PMSF,10 mM β-glycerolphosphate, 0.1 mM Na₃VO₄, 30 mM 4-nitrophenylphosphate)to give 1×10⁸ cells/17.5 ml. The cells are lysed for 30 minutes on ice.After removal of the cell debris by centrifugation (4000 rpm, 5 minutes)the clear supernatant is combined with glutathione sepharose beads (1 mlresuspended and washed beads per 50 ml of supernatant) and the mixtureis incubated for 30 minutes at 4° C. on a rotating board. Then the beadsare washed with lysis buffer and the recombinant protein is eluted fromthe beads with 1 ml elution buffer/ml resuspended beads (elution buffer:100 mM Tris/HCl pH=8.0, 120 mM NaCl, 20 mM reduced glutathione (SigmaG-4251), 10 mM MgCl₂, 1 mM DTT). The protein concentration is determinedby Bradford Assay.

Assay Procedure

The following components are combined in a well of a 96-wellround-bottomed dish (Greiner bio-one, PS Microtitre plate No. 650101):

-   -   10 μl of the compound to be tested in variable concentrations        (e.g. beginning at 300 μM, and dilution to 1:3) in 6% DMSO, 0.5        mg/ml casein (Sigma C-5890), 60 mM β-glycerophosphate, 25 mM        MOPS pH=7.0, 5 mM EGTA, 15 mM MgCl₂, 1 mM DTT    -   20 μl substrate solution (25 mM MOPS pH=7.0, 15 mM MgCl₂, 1 mM        DTT, 2.5 mM EGTA, 30 mM β-glycerophosphate, 0.25 mg/ml casein)    -   20 μl enzyme dilution (1:100 dilution of the enzyme stock in 25        mM MOPS pH=7.0, 15 mM MgCl₂, 1 mM DTT)    -   10 μl ATP solution (45 μM ATP with 1.11×10⁶ Bq/ml        gamma-P33-ATP).

The reaction is started by adding the ATP solution and continued for 45minutes at 30° C. with gentle shaking (650 rpm on an IKA shaker MTS2).The reaction is stopped by the addition of 125 μl of ice-cold 5% TCA perwell and incubated on ice for at least 30 minutes. The precipitate istransferred by harvesting onto filter plates (96-well microtitre filterplate: UniFilter-96, GF/B; Packard; No. 6005177), then washed four timeswith 1% TCA and dried at 60° C. After the addition of 35 μlscintillation solution (Ready-Safe; Beckmann) per well the plate issealed shut with sealing tape and the amount of P33 precipitated ismeasured with the Wallac Betacounter. The measured data are evaluatedusing the standard Graphpad software (Levenburg-Marquard algorithm).

Measurement of Cytotoxicity on Cultivated Human Tumour Cells

To measure cytotoxicity on cultivated human tumour cells, cells ofcervical carcinoma tumour cell line HeLa S3 (obtained from American TypeCulture Collection (ATCC)) were cultivated in Ham's F12 Medium (LifeTechnologies) and 10% foetal calf serum (Life Technologies) andharvested in the log growth phase. Then the HeLa S3 cells were placed in96-well plates (Costar) at a density of 1000 cells per well andincubated overnight in an incubator (at 37° C. and 5% CO₂), while oneach plate 6 wells were filled with medium alone (3 wells as the mediumcontrol, 3 wells for incubation with reduced AlamarBlue reagent). Theactive substances were added to the cells in various concentrations(dissolved in DMSO; DMSO final concentration: 0.1%) (in each case as atriple measurement). After 72 hours incubation 20 μl AlamarBlue reagent(AccuMed International) were added to each well, and the cells wereincubated for a further 7 hours. As a control, 20 μl reduced AlamarBluereagent was added to each of 3 wells (AlamarBlue reagent, which wasautoclaved for 30 min). After 7 h incubation the colour change of theAlamarBlue reagent in the individual wells was determined in a PerkinElmer fluorescence spectrophotometer (excitation 530 nm, emission 590nm, slits 15 nm, integration time 0.1 ms). The amount of AlamarBluereagent reacted represents the metabolic activity of the cells. Therelative cell activity was calculated as a percentage of the control(HeLa S3 cells without inhibitor) and the active substance concentrationwhich inhibited the cell activity by 50% (IC50) was derived. The valueswere calculated from the average of three individual measurements—withcorrection of the dummy value (medium control).

FACS Analysis

Propidium iodide (PI) binds stoichiometrically to double-stranded DNA,and is thus suitable for determining the proportion of cells in the G1,S, and G2/M phase of the cell cycle on the basis of the cellular DNAcontent. Cells in the G0 and G1 phase have a diploid DNA content (2N),whereas cells in the G2 or mitosis phase have a 4N DNA content.

For PI staining, for example, 0.4 million HeLa S3 cells were seeded ontoa 75 cm² cell culture flask, and after 24 h either 0.1% DMSO was addedas control or the substance was added in various concentrations (in 0.1%DMSO). The cells were incubated for 24 h with the substance or with DMSObefore the cells were washed 2× with PBS and then detached withtrypsin/EDTA. The cells were centrifuged (1000 rpm, 5 min, 4° C.), andthe cell pellet was washed 2× with PBS before the cells were resuspendedin 0.1 ml PBS. Then the cells were fixed with 80% ethanol for 16 hoursat 4° C. or alternatively for 2 hours at −20° C. The fixed cells (10⁶cells) were centrifuged (1000 rpm, 5 min, 4° C.), washed with PBS andthen centrifuged again. The cell pellet was resuspended in 0.25% TritonX-100 in 2 ml PBS, and incubated on ice for 5 min before 5 ml PBS wereadded and the mixture was centrifuged again. The cell pellet wasresuspended in 350 μl PI staining solution (0.1 mg/ml RNase A, 10 μg/mlpropidium iodide in 1×PBS). The cells were incubated for 20 min in thedark with the staining buffer before being transferred into samplemeasuring containers for the FACS scan. The DNA measurement was carriedout in a Becton Dickinson FACS Analyzer, with an argon laser (500 mW,emission 488 nm), and the DNA Cell Quest Programme (BD). The logarithmicPI fluorescence was determined with a band-pass filter (BP 585/42). Thecell populations in the individual cell cycle phases were quantifiedusing the ModFit LT Programme made by Becton Dickinson.

The compounds according to the invention were also tested accordinglyfor other tumour cells. For example, these compounds are effective oncarcinomas of many different kinds of tissue (e.g. breast (MCF7); colon(HCT116), head and neck (FaDu), lung (NCI-H460), pancreas (BxPC-3) andprostate (DU145)), sarcomas (e.g. SK-UT-1B), leukaemias and lymphomas(e.g. HL-60; Jurkat, THP-1) and other tumours (e.g. melanomas (BRO),gliomas (U-87MG)) and could be used for such indications. This isevidence of the broad applicability of the compounds according to theinvention for the treatment of many different kinds of tumour types.

The compounds of general formula (I) may be used on their own or inconjunction with other active substances according to the invention,optionally also in conjunction with other pharmacologically activesubstances. Suitable preparations include for example tablets, capsules,suppositories, solutions, particularly solutions for injection (s.c.,i.v., i.m.) and infusion, elixirs, emulsions or dispersible powders. Thecontent of the pharmaceutically active compound(s) should be in therange from 0.1 to 90 wt.-%, preferably 0.5 to 50 wt.-% of thecomposition as a whole, i.e. in amounts which are sufficient to achievethe dosage range specified below. The doses specified may, if necessary,be given several times a day.

Suitable tablets may be obtained, for example, by mixing the activesubstance(s) with known excipients, for example inert diluents such ascalcium carbonate, calcium phosphate or lactose, disintegrants such ascorn starch or alginic acid, binders such as starch or gelatine,lubricants such as magnesium stearate or talc and/or agents for delayingrelease, such as carboxymethyl cellulose, cellulose acetate phthalate,or polyvinyl acetate. The tablets may also comprise several layers.

Coated tablets may be prepared accordingly by coating cores producedanalogously to the tablets with substances normally used for tabletcoatings, for example collidone or shellac, gum arabic, talc, titaniumdioxide or sugar. To achieve delayed release or preventincompatibilities the core may also consist of a number of layers.Similarly the tablet coating may consist of a number or layers toachieve delayed release, possibly using the excipients mentioned abovefor the tablets.

Syrups or elixirs containing the active substances or combinationsthereof according to the invention may additionally contain a sweetenersuch as saccharine, cyclamate, glycerol or sugar and a flavour enhancer,e.g. a flavouring such as vanillin or orange extract. They may alsocontain suspension adjuvants or thickeners such as sodium carboxymethylcellulose, wetting agents such as, for example, condensation products offatty alcohols with ethylene oxide, or preservatives such asp-hydroxybenzoates.

Solutions for injection and infusion are prepared in the usual way, e.g.with the addition of isotonic agents, preservatives such asp-hydroxybenzoates, or stabilisers such as alkali metal salts ofethylenediamine tetraacetic acid, optionally using emulsifiers and/ordispersants, whilst if water is used as the diluent, for example,organic solvents may optionally be used as solvating agents ordissolving aids, and transferred into injection vials or ampoules orinfusion bottles.

Capsules containing one or more active substances may for example beprepared by mixing the active substances with inert carriers such aslactose or sorbitol and packing them into gelatine capsules. Suitablesuppositories may be made for example by mixing with carriers providedfor this purpose, such as neutral fats or polyethyleneglycol or thederivatives thereof.

Excipients which may be used include, for example, water,pharmaceutically acceptable organic solvents such as paraffins (e.g.petroleum fractions), vegetable oils (e.g. groundnut or sesame oil),mono- or polyfunctional alcohols (e.g. ethanol or glycerol), carrierssuch as e.g. natural mineral powders (e.g. kaolins, clays, talc, chalk),synthetic mineral powders (e.g. highly dispersed silicic acid andsilicates), sugars (e.g. cane sugar, lactose and glucose), emulsifiers(e.g. lignin, spent sulphite liquors, methylcellulose, starch andpolyvinylpyrrolidone) and lubricants (e.g. magnesium stearate, talc,stearic acid and sodium lauryl sulphate).

The preparations are administered by the usual methods, preferably byoral or transdermal route, most preferably by oral route. For oraladministration the tablets may, of course contain, apart from theabovementioned carriers, additives such as sodium citrate, calciumcarbonate and dicalcium phosphate together with various additives suchas starch, preferably potato starch, gelatine and the like. Moreover,lubricants such as magnesium stearate, sodium lauryl sulphate and talcmay be used at the same time for the tabletting process. In the case ofaqueous suspensions the active substances may be combined with variousflavour enhancers or colourings in addition to the excipients mentionedabove.

For parenteral use, solutions of the active substances with suitableliquid carriers may be used.

The dosage for intravenous use is from 1-1000 mg per hour, preferablybetween 5 and 500 mg per hour.

However, it may sometimes be necessary to depart from the amountsspecified, depending on the body weight, the route of administration,the individual response to the drug, the nature of its formulation andthe time or interval over which the drug is administered. Thus, in somecases it may be sufficient to use less than the minimum dose givenabove, whereas in other cases the upper limit may have to be exceeded.When administering large amounts it may be advisable to divide them upinto a number of smaller doses spread over the day.

In another aspect the present invention relates to pharmaceuticalformulations, preferably pharmaceutical formulations of the typedescribed above, characterised in that they contain one or morecompounds of general formula (I).

The formulation examples which follow illustrate the present inventionwithout restricting its scope:

EXAMPLES OF PHARMACEUTICAL FORMULATIONS

A) Tablets per tablet active substance 100 mg lactose 140 mg corn starch240 mg polyvinylpyrrolidone  15 mg magnesium stearate  5 mg 500 mg

The finely ground active substance, lactose and some of the corn starchare mixed together. The mixture is screened, then moistened with asolution of polyvinylpyrrolidone in water, kneaded, wet-granulated anddried. The granules, the remaining corn starch and the magnesiumstearate are screened and mixed together. The mixture is compressed toproduce tablets of suitable shape and size.

B) Tablets per tablet active substance  80 mg lactose  55 mg corn starch190 mg microcrystalline cellulose  35 mg polyvinylpyrrolidone  15 mgsodium-carboxymethyl starch  23 mg magnesium stearate  2 mg 400 mg

The finely ground active substance, some of the corn starch, lactose,microcrystalline cellulose and polyvinylpyrrolidone are mixed together,the mixture is screened and worked with the remaining corn starch andwater to form a granulate which is dried and screened. Thesodium-carboxymethyl starch and the magnesium stearate are added andmixed in and the mixture is compressed to form tablets of a suitablesize.

C) Ampoule solution active substance 50 mg sodium chloride 50 mg waterfor inj.  5 ml

The active substance is dissolved in water at its own pH or optionallyat pH 5.5 to 6.5 and sodium chloride is added to make it isotonic. Thesolution obtained is filtered free from pyrogens and the filtrate istransferred under aseptic conditions into ampoules which are thensterilised and sealed by fusion. The ampoules contain 5 mg, 25 mg and 50mg of active substance.

1. A compound of the formula